HIV-2 envelope polypeptides

ABSTRACT

Polypeptides which include at least one antigenic and/or immunogenic determinant of the envelope protein (env) of the HIV-2 virus and their production by recombinant DNA technology and the DNA sequences, expression vectors and transformed single-cell organisms used in this process are provided. A method for the detection of HIV antibodies or HIV viruses in human sera or in other biological fluids is also provided.

This is a division of application Ser. No. 08/213,416 filed Mar. 15,1994, which is a continuation of Ser. No. 07/895,977, filed Jun. 9,1992, abandoned, which is a continuation of Ser. No. 07/268,322, filedNov. 7, 1988, abandoned.

TECHNICAL FIELD

The present invention relates to novel polypeptides comprising at leastone antigenic and/or immunogenic determinant of the envelope protein(env) of the HIV-2 virus, DNA sequences which code for thesepolypeptides, recombinant vectors which contain these DNA sequences,microorganisms transformed with these recombinant vectors and processesfor the production of the polypeptides by recombinant DNA technology.The invention also relates to a method for the detection of HIVantibodies (HIV-2 antibodies or HIV-1 and HIV-2 antibodies) or HIVviruses (HIV-2 viruses or HIV-1 and HIV-2 viruses) in human serum orother biological body fluids.

BACKGROUND OF THE INVENTION

In 1986 a new virus designated as HIV-2 was isolated from West AfricanAIDS patients Clavel et al., Science 233, 343-346 (1986); Clavel et al.,Nature 324, 691-695 (1986)!. On the basis of its morphology, itslymphotropism and its cytophatic in vitro activity on T₄ -positivecells, this virus was associated with the AIDS-causing HIV-1 viruses. Inspite of these similiar properties, a genetic comparison of HIV-1 andHIV-2 viruses showed, however, only a limited sequence homology Guyaderet al., Nature 326, 662-669 (1987)!.

More than 20 different HIV-2 viruses have been isolated from WestAfrican AIDS patients and from European AIDS patients Guyader et al.,supra; Brun-Vezinet et al., Lancet 1, 128-132 (1987)!. The sera of theseAIDS patients were all negative in the HIV-1 ELISA Brun-Vezinet et al.,supra!. Accordingly, there is a very great need for a precise and rapidmethod for the diagnosis of HIV-2 viruses in human blood and in otherbody fluids.

SUMMARY OF THE INVENTION

Accordingly, novel polypeptides having amino acid sequences whichcorrespond to at least one antigenic and/or immunogenic determinant ofthe envelope protein (env) of the HIV-2 virus have been produced byusing recombinant DNA techniques. These polypeptides permit thedetection of HIV-2 antibodies or HIV-2 viruses or fragments thereof inhuman sera or other biological body fluids.

The present invention provides such polypeptides. More precisely, theinvention provides polypeptides having the amino acid sequence

    ______________________________________                                        SerAlaArgLeuAsnSerTrpGlyCysAlaPheArgGlnValCysHisThrThr                        ValProTrpValAsnAspSerLeuAlaProAspTrpAspAsnMetThrTrpGln                        GluTrpGluLysGlnValArgTyrLeuGluAlaAsnIleSerLysSerLeuGlu                        GlnAlaGlnGly (ENV(60))         (I)                                            ______________________________________                                    

or subsequences or functional equivalents thereof which correspond to atleast one antigenic and/or immunogenic determinant of the HIV-2 envprotein.

The invention also provides the previously defined polypeptides,fragments and functional equivalents which are additionally covalentlylinked to an affinity peptide and a carrier polypeptide. The previouslydefined polypeptides which are additionally covalently linked with anaffinity peptide and a polypeptide whose amino acid sequence correspondsto at least one antigenic and/or immunogenic determinant of the HIV-1envelope protein (env) and/or of the HIV-1 core protein (gag) are alsoprovided. Since such fusion proteins have antigenic and/or immunogenicdeterminants of both HIV-1 viruses and HIV-2 viruses, they can be usedas an effective diagnostic tool for the simultaneous detection of HIV-1and HIV-2 antibodies or HIV-1 and HIV-2 viruses or fragments thereof inhuman sera or other biological body fluids.

The preferred polypeptides of the present invention are defined by thegeneral formulae

    A--B--C

    A--C--B

and

    C--B--A

wherein

A is an affinity peptide,

B is a polypeptide having the amino acid sequence of formula I and

C is a carrier polypeptide or a polypeptide, the amino acid sequence ofwhich corresponds to at least one antigenic and/or immunogenicdeterminant of the HIV-1 envelope protein (env) and/or the HIV-1 coreprotein (gag). The latter polypeptide will hereinafter be referred to asan "HIV-1 polypeptide."

Especially preferred polypeptides are those having the formula ##STR1##

BRIEF DESCRIPTION OF THE FIGURES

The present invention can be more readily understood by reference to theaccompanying Figures, in which the following abbreviations and symbolsare used:

B, Bg, E, H, Sa, X and Xb denote cleavage sites for the restrictionenzymes BamHI, BglII, EcoRI, HindIII, PstI, SalI, XhoI and XbaI,respectively.

represents the promoters of the genes bla, lacI and neo; represents theribosomal binding sites of the genes bla. cat, neo and lacI; representsthe terminators t_(o) and T1; represents the regulatablepromoter/operator element N25OPSN25OP29; represents the ribosomalbinding site RBSII; → represents the coding region under control of thisribosomal binding site; represents the regions which code for the sixhistidines; represents the region which is required for the replication(repl.); represents coding regions for dihydrofolate reductase (dhfr),chloramphenicol acetyltransferase, lac repressor (lacI), β-lactamase(bla) and neomycin phosphotransferase (neo); ▭ represents the syntheticenv(60) gene;  represents HIV-1 gene fragments.

The contents of the figures can be summarized as follows:

FIG. 1 Representation of the nucleotide sequence of the syntheticenv(60) gene and the amino acid sequence of the ENV(60) polypeptidederived therefrom. The individual oligonucleotide fragments from whichthe synthetic env(60) gene is constructed are denoted by the numbers1-14.

FIG. 2 Schematic representation of plasmid pDS78/RBSII.

FIG. 3 Nucleotide sequence of the plasmid pDS78/RBSII. In the sequencethe recognition sites for the restriction enzymes set forth in FIG. 2are overlined, while the regions coding for β-lactamase anddihydrofolate reductase are underlined.

FIG. 4 Schematic representation of plasmid pDMI,1.

FIG. 5 Nucleotide sequence of plasmid pDMI,1. In the sequence therecognition sites for the restriction enzymes set forth in FIG. 4 areoverlined, while the regions coding for neomycin phosphotransferase andlac repressor are underlined.

FIG. 6 Schematic representation of the production of the XhoI/BamHIfragment containing the regulatable promoter/operator elementN25OPSN25OP29, the ribosomal binding site RBSII and the region codingfor six histidines.

FIG. 7 Schematic representation of the construction of plasmidpDS78/RBSII,6xHis using the plasmid pDS78/RBSII and the XhoI/BamHIfragment containing the regulatable promoter/operator elementN25OPSN25OP29, the ribosomal binding site RBSII and the region codingfor six histidines.

FIG. 8 Schematic representation of the construction of plasmidpenv(60)-DHFR.

FIG. 9 Schematic representation of the construction of plasmidpDHFR-env(80).

FIG. 10 Reactivity of the ENV(60)-DHFR and DHFR-ENV(60) polypeptideswith HIV-positive sera.

The upper part shows the electrophoretic analysis of E. coli M15 lysatescontaining plasmids pDS78/RSBII,6xHis (trace a), penv(60)-DHFR (trace b)and pDHFR-env(60) (trace c). Trace d contains purified HIV-1ENV(80)-DHFR. The lower part shows the corresponding immunoblots whichwere produced with HIV-2 (left) and HIV-1 (right) positive serum. The Mtraces contain pre-stained molecular weight standards, the sizes ofwhich are given in kilodaltons.

FIG. 11 Schematic representation of plasmid pDS56/RBSII.

FIG. 12 Nucleotide sequence of plasmid pDS56/RBSII. The cleavage sitesfor restriction enzymes set forth in FIG. 11 are overlined, while theregion under control of the RBSII is underlined.

FIG. 13 Schematic representation of the construction and isolation ofthe BamHI/XbaI fragment containing the region coding for 6 histidines,the terminator t_(o), the cat gene and the terminator T1, which was usedin the construction of plasmid pRBSII-6xHis.

FIG. 14 Schematic representation of the construction of plasmidpRBSII-6xHis by linking the BamHI/XbaI fragment shown in FIG. 13 withthe replication region-containing XbaI/BamHI fragment of plasmidpDS56/RBSII.

FIG. 15 Schematic representation of the production of the HIV-1BamHI/BglII-gag(419) gene fragment.

FIG. 16 Representation of the nucleotide sequence of the HIV-1 gag genefragment present in plasmid pU-GAG.

FIG. 17 Representation of the nucleotide sequence of the HIV-1 gag genefragment present in plasmid p2-3U/HindIII 10.

FIG. 18 Schematic representation of the construction of plasmidpRBSII-gag(419)-6xHis.

FIG. 19 Schematic representation of the construction of plasmidpRBSII-env(80)-gag(419)-6xHis.

FIG. 20 Schematic representation of the construction of plasmidpRBSII-env(80)-gag(419)-env(60)-6xHis.

DESCRIPTION OF THE INVENTION

The term "functional equivalent" which is used in connection with thepolypeptides of the invention relates to polypeptides whose amino acidsequences have been derived from the amino acid sequences indicatedabove by nucleotide substitutions, deletions, insertions or additionsand which correspond to at least one antigenic and/or immunogenicdeterminant of the HIV-2 env protein.

Certain substitutions in the amino acid sequence of a polypeptide haveno influence on the biological activity of a polypeptide. Examples ofsuch amino acid substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and vice versa (see Doolittle, in"The Proteins", ed. Neurath, H, and Hill, R. L., Academic Press, NewYork 1979!).

As used herein, the term "affinity peptide" means a peptide whichcontains an amino acid sequence which preferably binds to an affinitychromatographic carrier material. Examples of such affinity peptides arepeptides which contain at least two adjacent histidine residues (see inthis respect European Patent Application Publication No. 282 042). Suchaffinity peptides bind selectively to nitrilotriacetic acid-nickelchelate resins Hochuli and Dobeli, Biol. Chem. Hoppe-Seyler 368, 748(1987); European Patent Application Publication No. 253 303!.Polypeptides which contain such an affinity peptide can therefore beseparated selectively from the remaining polypeptides. The affinitypeptide can be linked either to the C-terminus or the N-terminus of thepolypeptides having the amino acid sequence of formula I or subsequencesor functional equivalents thereof.

The term "carrier polypeptide" which is used in connection with thepolypeptides of the invention relates to such polypeptides whichthemselves contain no antigenic and/or immunogenic determinants of theHIV-2 env protein, but which are required for the expression of thepolypeptides having the amino acid sequence of formula I or fragments orfunctional equivalents thereof. Preferred carrier polypeptides are E.coli chloramphenicol acetyltransferase (CAT) and mouse dihydrofolatereductase (DHFR).

Because of the process for their production, the polypeptides of theinvention may contain methionine (coded for by ATG) as the firstN-terminal amino acid. Alternatively, the microbial host may process thetranslation product partially or completely, whereby the N-terminalmethionine is cleaved off.

The polypeptides of the invention can also be present in the form ofmultimers, e.g., in the form of dimers, trimers, tetramers etc. Ofcourse, the polypeptides can also be covalently linked additionally withpolypeptides whose amino acid sequence corresponds to at least oneantigenic and/or immunogenic determinant of the HIV-2 core protein(gag).

The invention also provides DNA sequences which code for thepolypeptides of the invention, recombinant vectors which contain theseDNA sequences, single-cell organisms containing such vectors for theproduction of the polypeptides and processes for the production of suchDNA sequences, recombinant vectors and single-cell organisms. Methodsfor the expression, isolation and purification of the polypeptides arealso described. The thus-produced polypeptides can be used for a numberof important immunological procedures, using the methods of thisinvention.

The polypeptides of the invention can be used as diagnostic reagents forthe detection of antibodies against HIV-2 viruses in human sera or forthe simultaneous detection of antibodies against HIV-1 and HIV-2 viruses(referred to hereinafter collectively as antibodies against HIV viruses)in human sera. Since they can be produced in homogeneous form, problemswith nonspecific reactions, which in the past have limited the use ofdiagnostic reagents based on relatively crude viral HIV protein lysates,can be eliminated.

When used as an immunogen, the polypeptides of the invention can be usedin animals to produce antibodies which are directed against theantigenic determinants contained in these polypeptides. Such antibodiescan be used, in turn, in combination with the polypeptides, which havebeen appropriately labeled, in a radioimmunoassay (RIA) or in an enzymeimmunoassay (ELISA) to detect the presence of HIV-2 viruses or HIV-1 andHIV-2 viruses (referred to hereinafter collectively as HIV viruses) orparticles thereof in human sera or in other biological fluids such as,e.g., in tears, semen, vaginal secretions and saliva. The particles (orfragments) of HIV viruses which can be detected using these methodsinclude, of course, pieces of the viral HIV envelope proteins (env).

The polypeptides of the invention can be produced using conventionalmethods of peptide synthesis in the liquid phase or, preferably, on thesolid phase, such as the methods of Merrifield (J. Am. Chem. Soc. 85,2149-2154 1963!), or by other equivalent methods known in the art.

Alternatively, the polypeptides can be produced using methods of DNArecombinant technology Maniatis et al. in "Molecular Cloning--ALaboratory Manual", Cold Spring Harbor Laboratory (1982), herebyincorporated by reference!. For example, the DNA sequences which codefor the polypeptides of the invention can be synthesized by conventionalchemical methods, e.g., by the phosphotriester method Narang et al., inMeth. Enzymol. 68, 90-108 (1979)! or by the phosphodiester method Brownet al., Meth. Enzymol. 68, 109-151 (1979)!. In both methods longoligonucleotides are usually synthesized which are joined to one anotherin the aforementioned manner. The nucleotide sequences of the DNAfragments can be identical with those nucleotide sequences which codefor the natural HIV-2 or HIV-1 and HIV-2 polypeptides. Since the geneticcode is degenerate, however, it will be understood that partially orcompletely different nucleotide sequences can also code for the samepolypeptides. If desired, there can be selected for the nucleotidesequences those codons which are also preferably used by the hostorganism for the expression of the polypeptide Grosjean et al., Gene 18,199-209 (1982)!. Care must be taken, however, to ensure that thethus-obtained DNA sequences do not contain partial sequences which makethe construction of the expression sectors difficult, e.g., byintroducing an undesired restriction enzyme cleavage site.

The DNA sequences which code for the polypeptides of the invention canalso be produced by isolating a DNA fragment which codes for the aminoacid sequence of formula I from isolated proviral HIV-2 DNA or fromgenomic DNA of cells in which the proviral HIV-2 genome has beenintegrated, and subsequently incorporating the fragment into a suitablevector which codes for the partial sequences A and C of the generalformulae A--B--C, A--C--B and C--B--A.

After the production of the DNA sequences which code for thepolypeptides of the invention, the sequences can be incorporated usingknown methods into any suitable expression vector which produces therequisite expression signals. Suitable vectors can be constructed fromsegments of chromosomal, non-chromosomal and synthetic DNA sequencessuch as, e.g., various known plasmids and phage DNA's. Examples of suchvectors can be found in the aforementioned textbook of Maniatis et al.Especially suitable vectors are plasmids of the pDS family Bujard etal., Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc.,Vol. 155, 416-433 (1987)!.

In the preferred embodiment of the present invention, a synthetic BamHlfragment which codes for the amino acid sequence of formula I (env(60)gene) was fused with BamHl or BglII-cleaved DNA of plasmidpDS78/RBSII,6xHis and with BglII-cleaved DNA of plasmidpRBSII-env(80)-gag(419)-6xHis, to isolate the expression vectorspenv(60)-DHFR. pDHFR-env(60) and pRBSII-env(80)-gag(419)-env(60)-6xHiswhich code for the synthesis of the especially preferred polypeptides offormulae II-IV of the invention. The synthesis of the synthetic env(60)gene is described in Example 1. Its nucleotide sequence and the aminoacid sequence (ENV(60)) derived therefrom are shown in FIG. 1. Theconstructions of plasmids pDS78/RBSII,6xHis, penv(60)-DHFR,pDHFR-env(60), pRBSII-env(80)-gag(419)-6xHis andpRBSII-env(80)-gag(419)-env(60)-6xHis are described in detail inExamples 2-4, 6 and 7.

Expression vectors which contain the DNA sequences coding for thepolypeptides of the invention operatively linked with an expressioncontrol sequence can be incorporated using conventional methods into anysuitable host organism. The selection of a suitable host organism isdetermined by different factors which are well known in the art. Thus,for example, compatibility with the chosen vector, toxicity of theexpression product, expression characteristics, necessary biologicalsafety precautions and costs play a role and a compromise between all ofthese factors must be found.

Suitable host organisms include gram-negative and gram-positivebacteria, for example E. coli and B. subtilis strains. Especiallypreferred host organism of the present invention are E. coli strain M15(described as OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-4741974!) and E. coli W3110 (ATCC No. 27325). In addition to theaforementioned E. coli strain, however, other generally accessible E.coli strains such as E. coli 294 (ATCC No. 31446) and E. coli RR1 (ATCCNo. 31343) can also be used.

The manner in which the expression of the polypeptides of the inventionis carried out depends on the chosen expression vector/host cell system.Usually, the host organisms which contain a desired expression vectorare grown under conditions which are optimal for the growth of the hostorganisms. At the end of the exponential growth, when the increase incell number per unit time decreases, the expression of the desiredpolypeptide is induced, i.e. the DNA coding for the desired polypeptideis transcribed and the transcribed mRNA is translated. The induction canbe carried out by adding an inducer or a derepressor to the growthmedium or by altering a physical parameter, e.g., a change intemperature. In the expression vectors used in the preferred embodimentsof the present invention, the expression is controlled by the lacrepressor. By adding isopropyl-β-D-thiogalactopyranoside (IPTG), theexpression control sequence is derepressed and the synthesis of thedesired polypeptide is thereby induced.

For the isolation of small amounts of the polypeptides of the inventionfor analytical purposes, e.g., for polyacrylamide gel electrophoreies,the host organisms can be disrupted by treatment with a detergent, e.g.,sodium dodecyl sulphate (SDS). Larger quantities of the polypeptides canbe recovered by mechanical Charm et al., Meth, Enzymol. 22, 476-556(1971)!, enzymatic (lysozyme treatment) or chemical (detergenttreatment, urea or guanidinium chloride treatment, etc.) means or by acombination of these means.

After the polypeptides of the invention have been removed from the hostorganisms they can be purified by known methods, e.g., by centrifugationat different velocities, precipitation with ammonium sulphate, dialysis(at normal pressure or at reduced pressure), preparative isoelectricfocusing, preparative gel electrophoresis or by various chromatographicmethods such as gel filtration, high performance liquid chromatography(HPLC), ion exchange chromatography, reverse phase chromatography andaffinity chromatography (e.g., on Sepharose® Blue C1-6B or oncarrier-bound monoclonal antibodies which are directed against thepolypeptides of the invention). Preferably, the polypeptides of theinvention are purified on nitrilotriacetic acid (NTA) resins of thegeneral formula Carrier matrix-Spacer-NH--(CH₂)_(x) --CH(COOH--N(CH₂COO⁻)₂ Ni²⁺ in which x signifies 2, 3 or 4. suitable carrier matriciesinclude materials which are used in affinity and gel chromatography, forexample cross-linked dextrans, agarose (especially in the form knownunder the trademark Sepharose®) or polyacrylamides. Useful spacersinclude the spacer groups which are well known in affinitychromatography, with the groups --CH₂ --CH(OH)--CH₂ -- and --O--CO--being preferred.

An especially preferred NTA resin for the purification of thepolypeptides has the formula

     Sepharose®CL 6B!--O--CH.sub.2 --CH(OH)--CH.sub.2 --NH--(CH.sub.2).sub.4 CH(COOH)--N(CH.sub.2 COO.sup.-).sub.2 Ni.sup.2+.

As mentioned previously, the polypeptides of the invention which areobtainable using the previously described methods can be used as adiagnostic tool for the detection of antibodies against HIV viruses inhuman sera. To this end, the polypeptides of the invention can be usedin numerous, known detection methods.

For example, the polypeptides can be labelled using known methods andthese labelled polypeptides can then be added to a human serum samplesuspected to contain antibodies against HIV viruses to form labelledpolypeptide/antibody complexes. The complexes thus formed can then bedetected using conventional methods.

The polypeptides of the invention can also first be immobilized on asolid support and then brought into contact with a human serum sample.Antibodies against HIV viruses in the sample bind to this immobilizedpolypeptide and, after removing non-bond polypeptides and antibodies bywashing, the thus-formed complexes can be detected using a reagent suchas Staphylococcus aureus protein A (labelled, for example, with ¹²⁵iodine) or a second anti-Ig antibody (labelled, for example, with aradioisotope or with horseradish peroxidase). Many modifications andvariations of these detection methods will be apparent to the personskilled in the art, of which some are suggested hereinafter.

Different diagnostic tests for the detection of HIV viruses or fragmentsthereof in human sera or in other biological fluids can be developed byusing antibodies against the polypeptides of the invention (hereinafteranti-HIV antibodies). Such antibodies can be produced by injecting animmunogenic composition containing a polypeptide of the invention and aphysiologically compatible carrier material into a mammal or bird. Theamount of protein required for the injection is known to the personskilled in the art or can be determined by routine experimentation usingknown methods. The term "carrier material" used in connection with thepresent invention relates either to known compositions which aresuitable for administration to human beings or to known adjuvants whichare used in the inoculation of animals.

Suitable adjuvants for use in human beings and animals are well known inthe art WHO Techn. Rep. Series 595, 1-400 (1976); Jollis et al.,"Chemical and Biological Basis of Adjuvants", in Molecular Biochemistryand Biophysics Vol. 13, 1-148 (1973) Springer Verlag Berlin!. Thepolypeptides of the invention can also be administered afterincorporation in liposomes or other micro-carrier materials, or aftercoupling to polysaccharides, other polypeptides or other polymers.

One or more additional inoculations (boosters) are generallyadministered a few weeks after the first inoculation, to produce a hightiter of anti-HIV antibodies. These can then be isolated using standardmethods.

Of course, monoclonal antibodies can also be used for the aforementionedtests. The methods for the production of such antibodies are well knownin the art Kohler et al., Nature 256, 495-497 (1975)!.

As explained previously, the anti-HIV antibodies can be used in variousdiagnostic tests for the detection of HIV viruses or fragments thereof.Such tests can be carried out in the form of radioimmunoassays, eitherin solution or on a solid support. However, enzyme immunoassays can alsobe carried out. The tests can be carried out either directly orindirectly by means of a second antibody which is directed against theanti-HIV antibody. Numerous enzyme activities can be coupled to theantibodies, e.g., peroxidase, glucose oxidase, β-galactosidase andalkaline phosphatase, which produce a coloration after the addition of asubstrate solution.

The principle underlying many of these tests is that human serum orother biological fluids suspected to contain HIV viruses or fragmentsthereof is/are reacted with a known titer of anti-HIV antibodies to formantigen/antibody complexes. The complexes thus formed are detected usingconventional methods.

It will be evident to those skilled in the art that many other detectionmethods employing the anti-HIV antisera can also be used, such asvarious agglutination tests. In agglutination tests, the interactionbetween antibodies and HIV viruses or fragments thereof is detectedusing particles coated with anti-HIV antibodies. Such particles are, forexample, latex beads, liposomes, erythrocytes, polyacrylamide beads orany of a number of suitable polymers.

The previously described methods for the detection of HIV viruses orantibodies against HIV viruses can be carried out using suitable testkits consisting of a container which contains a polypeptide of theinvention or anti-HIV antibodies of the present invention.

EXAMPLES

The following, nonlimiting examples will further serve to illustratethis invention. Unless otherwise specified, percentages given below forsolids in solid mixtures, liquids in liquids and solids in liquids areon a wt/wt, vol/vol and wt/vol basis, respectively.

EXAMPLE 1 Production of the Synthetic HIV-2 Env(60) Gene

A) Principles

The synthetic HIV-2 env(60) gene was constructed from 14 oligonucleotidefragments, the length of which varied between 17 and 31 nucleotides (seeFIG. 1).

B) Synthesis of the Oligonucleotide Fragments

The oligonucleotide fragments 1-14 were synthesized simultaneously onsolid carrier material using the procedure described by Bannwarth andIaiza in DNA 5, 413-419 (1986).

C) Combination of the oligonucleotide Fragments

100 pmol of each of the oligonucleotide fragments 2, 3, 8, 9, 10 and,respectively, 4, 5, 6, 7, 11, 12, 13 were phosphorylated at 37° C. for15 minutes in 100 μl of kinase buffer Maniatis et al., "MolecularCloning", Cold Spring Harbor Laboratory (1982)! containing 100 units ofpolynucleotide kinase and 100 μCi of γ-³² P-ATP (5000 Ci/mmol).Thereafter, a 10-fold excess of cold ATP was added to the reactionmixtures. After a further incubation period of 90 minutes, thepolynucleotide kinase was heat-inactivated (2 minutes, 95° C.) and,after the addition of 10 μl of 5M lithium acetate (LiOAc) and 100 μl ofisopropanol, the phosphorylated oligonucleotide fragments wereprecipitated for 30 minutes at -78° C. The precipitated oligonucleotidefragments were then washed with ethanol and dried.

The phosphorylated oligonucleotide fragments 2, 3, 8, 9, 10 and thenon-phosphorylated fragments 1 and, respectively, the phosphorylatedoligonucleotide fragments 4, 5, 6, 7, 11, 12, 13 and thenon-phosphorylated fragment 14 were hybridized according to knownmethods described in the literature Maniatis et al., supra! and thenligated (37° C., 1.5 hours) in 100 μl of ligase buffer Maniatis et al.,supra! containing 31 units of T4 ligase. Subsequently, the twosub-fragments obtained were precipitated as previously described, thenwashed with ethanol and dried.

The two sub-fragments were subsequently separated by 6% polyacrylamidegel electrophoresis, eluted according to standard methods Maniatis etal., supra!, freed from salts by using a Sephadex G-50 column and linkedwith one another by using T4 ligase as previously described, to give thedesired HIV-2 env(60) gene. After purification of the HIV-2 env(60) geneby the previously described procedure (polyacrylamide gelelectrophoresis, elution and removal of salts), the gene wasphosphorylated as previously described.

EXAMPLE 2 Construction of Plasmid pDS78/RBSII,6xHis

A. Description of Plasmids pDS78/RBSII and pDMI,1

For the construction of plasmid pDS78/RBSII,6xHis plasmid pDS78/RBSIIwas chosen. E. coli cells transformed with this plasmid and with plasmidpDMI,1 have been deposited under the Budapest Treaty at the DeutschenSammlung von Mikroorganismen in Gottingen on the Sep. 3, 1987 E. coliM15 (pDS78/RBSII, pDMI,1), DSM No. 4232!.

The part of pDS78/RBSII (FIGS. 2 and 3) which lies between therestriction cleavage sites for XbaI and XhoI and which contains thereplication region and the gene for β-lactamase, which confersampicillin resistance to the cells, was derived originally from theplasmid pBR322 Bolivar et al., Gene 2, 95-113 (1977); Sutcliffe, ColdSpring Harbor Symp. Quant. Biol. 43, 77-90 (1979)!. However, the genefor β-lactamase was modified so that the cleavage sites for therestriction enzymes HincII and PstI were eliminated. These alterationsin the DNA sequence did not, however, affect the amino acid sequence ofthe β-lactamase.

The remaining part of the plasmid carries the regulatablepromoter/operator element- N25OPSN25OP29 and the ribosomal binding siteRBSII. This ribosomal binding site was derived from the ribosomalbinding site of the promoter P_(G25) of the E. coli phage T5 R. Gentz,Thesis, University of Heidelberg, BRD (1984)! and was obtained as anEcoRI/BamHI fragment by DNA synthesis. This is followed by thedihydrofolate reductase gene of the mouse cell line AT-3000 Chang etal., Nature 275, 617-624 (1978); Masters et al., Gene 21, 59-63 (1983)!,which was altered by introducing a cleavage site for the restrictionenzyme BglII directly in front of the termination codon for translation.

Plasmid pDS78/RBSII also contains the terminator t_(o) of the E. coliphage lambda Schwarz et al., Nature 272, 410-414 (1978)!, thepromoter-free gene of chloramphenicol acetyltransferase Marcoli et al.,FEBS Letters, 110, 11-14 (1980)! and the terminator T1 of the E. colirrnB operon Brosius et al., J. Mol. Biol., 148, 107-127 (1981)!.

Plasmid pDS78/RBSII contains the regulatable promoter/operator elementN25OPSN25OP29 and the ribosomal binding site RBSII. Because of the highefficiency of this expression signal, plasmid pDS78/RBSII andderivatives thereof such as plasmid pDS78/RBSII,6xHis can be stablymaintained in E. coli cells only when the promoter/operator element isrepressed by the binding of a lac repressor to the operator. The lacrepressor is coded by the lacI gene. N25OPSN25OP29 can be repressedefficiently only when a sufficient number of repressor molecules arepresent in the cells. Therefore, the lacI^(q) allele, which contains apromoter mutant that produces an increased expression of the repressorgene, was used.

This lacI^(q) allele is contained in the plasmid pDMI,1 (FIGS. 4 and 5).This plasmid carries, in addition to the lacI gene, the neo gene whichconfers kanamycin resistance to the bacteria and which is used as aselection marker. pDMI,1 is compatible with the above-mentionedplasmids. E. coli cells which are transformed with such expressionvectors must contain pDMI,1 to guarantee that the expression vector ismaintained stable in the cells. Induction of this system is achieved byadding IPTG to the medium at the desired cell density.

Plasmid pDMI,1 (FIGS. 4 and 5) carries the neo gene of neomycinphosphotransferase from the transposon Tn5 Beck et al., Gene 19, 327-336(1982)!, which confers kanamycin resistance to the E. coli cells, andthe lacI gene Farabough, Nature 274, 765-769 (1978)! having the promotermutation I^(q) Calos, Nature 274, 762-765 (1978)!, which codes for thelac repressor. Moreover, the plasmid pDMI,1 contains a region of plasmidpACYC184 Chang et al., J. Bacteriol. 134, 1141-1156 (1978)! whichcontains all of the information required for replication and stabletransmission to the daughter cells.

B. Construction of Plasmid pDS78/RBSII,6xHis

For the construction of plasmid pDS78/RBSII,6xHis (FIGS. 6 and 7), theEcoRI/BamHI fragment of plasmid pDS78/RBSII comprising the ribosomalbinding site RBSII was combined with a region coding for six histidines.

For this purpose, two complementary oligonucleotides, the nucleotidesequences of which are represented in FIG. 6 as a double-stranded DNAsequence, were synthesized as previously described (Ex. 1, section B).The lyophilized oligonucleotides were taken up in water and dissolved at4° C. for 1 hour. The DNA concentration was 100 nmol/ml.

For the phosphorylation, 150 pmol of each of the two oligonucleotides in20 μl of 50 mM Tris/HCl pH 85! and 10 mM MgCl₂ were incubated at 37° C.for 20 minutes with 2 pmol of γ- ³² P!-ATP (5000 Ci/mmol) and 1 unit (U)of T4-polynucleotide kinase. Subsequently, 5 nmol of ATP were added and,after a further 20 minutes at 37° C., the reactions were terminated byheating to 65° C.

The DNA of the plasmid pDS78/RBSII was prepared for ligation with thetwo phosphorylated oligonucleotides by first cleaving 2 pmol of theplasmid DNA with the restriction enzyme BamHI. The DNA was extractedwith phenol, washed with ether and then precipitated using lithiumacetate/isopropanol as previously described. The sediment was dried andtaken up in 20 μl of TE buffer.

For the ligation with the phosphorylated oligonucleotides, 1.5 pmol ofthe plasmid DNA cleaved with BamHI were incubated at 15° C. for 2 hourswith 60 pmol of each of the phosphorylated oligonucleotides in ligasebuffer containing 2 units of T4-DNA ligase. After an incubation at 65°C. for 5 minutes, the ligated DNA was cleaved with the restrictionenzymes XhoI and BamHI. Thereafter, the XhoI/BamHI fragment containingthe regulatable promoter N25OPSN25OP29, the ribosomal binding site RBSIIand the region coding for 6 histidines (FIG. 6) was isolated by agarosegel electrophoresis.

For the construction of plasmid pDS78/RBSII,6xHis, the above XhoI/BamHIfragment was integrated into plasmid pDS78/RBSII, whereby the originalXhoI/BamHI fragment of this plasmid was replaced (FIG. 7). For thispurpose, 1 pmol of DNA of plasmid pDS78/RBSII was cleaved with therestriction enzymes XhoI and BamHI, before the larger DNA fragment wasisolated by agarose gel electrophoresis. 0.05 pmol of this fragment werethen incubated at 15° C. for 2 hours with 0.1 pmol of the isolatedXhoI/BamHI fragment in ligation buffer containing 2 units of T4-DNAligase.

E. coli M15 cells containing plasmid pDMI,1 were prepared for thetransformation by the method of Morrison Methods Enzymol. 68, 326-331(1979)!. After heating to 65° C. for 7 minutes the ligation mixture wasadded to 200 μl of these competent cells. The sample was held in ice for30 minutes, incubated at 42° C. for 2 minutes and, after the addition of0.5 ml of LB medium, incubated at 37° C. for 90 minutes.

The transformed cells were then plated onto LB agar plates containing100 μg/ml ampicillin and 25 μl/ml kanamycin, and the plates wereincubated at 37° C. overnight. Individual colonies were picked with asterile toothpick, transferred into a test tube which contained 10 ml LBmedium containing 100 μl/ml ampicillin and 25 μg/ml kanamycin andincubated for 12 hours in a shaking incubator. Thereafter, the cellswere sedimented and the plasmid DNA was isolated using the method ofBirnboim and Doly Nucleic Acids Res. 7, 1515-1523 (1979)!. 0.2 μg ofeach of the isolated plasmids were cleaved with the restriction enzymesXhoI and BamHI to determine whether, a fragment which contains theregulatable operator/repressor element N25OPSN25OP29, the ribosomalbinding site RBSII and the region coding for the six histidines waspresent in these plasmids. Plasmids containing such a fragment weredesigned pDS78/RBSII,6xHis (FIG. 7).

To demonstrate that the correct sequence was present inpDS78/RBSII,6xHis, the double-stranded circular plasmid DNA wassequenced, using a γ- ³² P!-ATP labelled starter sequence (primer). Thisstarter sequence contained the nucleotides of position 89-108 of plasmidpDS78/RBSII. 0.3 pmol of the isolated plasmid DNA were precipitated withalcohol, the sediment was washed once with 80% ethanol, dried andfinally dissolved in 8 μl of 1/4 TE buffer. The sample was incubated at95° C. for 5 minutes, cooled to 0° C. and centrifuged (Eppendorf benchcentrifuge, 2 minutes, 12,000 rpm). 1.5 pmol of the starter sequence ina volume of 2 μl were added before the sample was incubated first at 95°C. for 2 minutes and then at 42° C. for 5 minutes. The DNA was sequencedusing the method of Sanger et al. Proc. Natl. Acad. Sci. USA 74,5463-6567 (1977)!. Because a radioactively labelled "primer" was used,all reactions were carried out with unlabelled deoxynucleotidetriphosphates. The DNA sequence analysis indicated thatpDS78/RBSII,6xHis contained the sequence given in FIG. 6.

EXAMPLE 3 Construction of Plasmid penv(60)-DHFR

A) Principles

For the construction of plasmid penv(60)-DHFR, plasmid pDS78/RBSII,6xHislinearized with the restriction enzyme BamHI was linked with thesynthetically produced env(60) gene (FIG. 8).

B) Preparation of Plasmid pDS78/RBSII,6xHis Linearized with BamHI(Fragment 1)

4 pmol of plasmid pDS78/RBSII were cleaved with the restriction enzymeBamHI. Subsequently, the DNA was treated with CIP calf intestinalphosphatase!. The enzymes were then heat-inactivated and, after theaddition of sample buffer, the DNA was separated in a 1% low meltagarose gel. After staining with ethidium bromide, the corresponding DNAband was cut out under UV light (300 nm) and the DNA was extracted usingstandard methods Maniatis et al., supra!.

C) Preparation of the HIV-2 Env(60) Gene (Fragment 2)

The preparation of this gene is described in Example 1.

D) Production of Plasmid penv(60)-DHFR

0.05 pmol of Fragment 1 and 0.1 pmol of Fragment 2 were incubated (15°C., 2 hours) with 1U of T4 ligase. After heat inactivation of theenzyme, the DNA was transformed into the E. coli strain M15 containingplasmid pDMI,1. The cells were plated onto LB agar plates containing 100μg/ml ampicillin and 25 μg/ml kanamycin. The plates were incubated at37° C. for 15 hours. The ligation gave about 500 colonies. Individualcolonies were each transferred into 10 ml of LB medium and grown at 37°C. overnight, and their plasmid DNAs were subsequently isolated usingstandard methods Maniatis et al., supra!.

E) Sequence Analysis of the HIV-2 Env(60) Gene Integrated into PlasmidpDS78/RBSII,6xHis

To demonstrate that the correct HIV-2 env(60) gene sequence was presentin the correct orientation in the BamHI site of plasmidpDS78/RBSII,6xHis, the double-stranded circular plasmid DNA wassequenced, using a starter sequence (primer) labelled with γ-³² P!-ATP.

0.3 pmol of the isolated plasmid DNA were precipitated with alcohol, thesediment was washed once with 80% ethanol, dried and finally dissolvedin 8 μl of 1/4 TE buffer. After the addition of 2 pmol of theradioactively labelled starter sequence, the sample was incubated firstat 95° C. for 2 minutes and then at 42° for 5 minutes. The DNA was thensequenced by the method of Sanger et al. Proc. Natl. Acad. Sci. USA 74,5463-6567 (1977)!. The sequence analysis indicated that the correctHIV-2 env(60) gene sequence had been integrated into the BamHIrestriction site of plasmid pDS78/RBSII,6xHis.

EXAMPLE 4 Construction of Plasmid pDHFR-env(60)

A) Principles

For the construction of plasmid pDHFR-env(60), plasmid pDS78/RBSII,6xHislinearized with the restriction enzyme BglII was linked with thesynthetically produced env(60) gene (FIG. 9).

B) Preparation of BglII-linearized Plasmid pDS78/RBSII, 6xHis (Fragment1)

4 pmol of plasmid pDS78/RBSII, 6xHis were cleaved with the restrictionenzyme BglII. Subsequently, the DNA was treated with CIP. The enzymeswere then heat-inactivated and, after the addition of sample buffer, theDNA was separated in a 1% low melt agarose gel. After staining withethidium bromide, the corresponding DNA band was cut out under UV light(300 nm) and then DNA was extracted using standard methods Maniatis etal., supra!.

C) Preparation of the HIV-2 Env(60) Gene (Fragment 2)

The preparation of this gene is described in Example 1.

D) Production of Plasmid pDHFR-env(60)

0.05 pmol of Fragment 1 and 0.1 pmol of Fragment 2 were incubated (15°C. 2 hours) with 1U of T4 ligase. After heat inactivation of the enzyme,the DNA was transformed into E. coli strain M15 which contained theplasmid pDMI,1. The cells were plated onto LB agar plates containing 100μg/ml ampicillin and 25 μg/ml kanamycin. The plates were incubated at37° C. for 15 hours. The ligation gave about 500 colonies. Individualcolonies were grown at 37° C. overnight in 10 ml of LB medium and theirplasmid DNAs were subsequently isolated using standard methods Maniatiset al., supra!.

E) Sequence Analysis of the HIV-2 Env(60) Gene Integrated into PlasmidpDS78/RBSII,6xHis

To demonstrate that the correct HIV-2 env(60) gene sequence was presentin the correct orientation in the BglII site of plasmidpDS78/RBSII,6xHis, the double-stranded circular plasmid DNA wassequenced, using a starter sequence (primer) labelled with γ-³² P!-ATP.

0.3 pmol of the isolated plasmid DNA was precipitated with alcohol, thesediment was washed once with 80% ethanol, dried and finally dissolvedin 8 μl of 1/4 TE buffer. After the addition of 2 pmol of theradioactively labelled primer, the sample was incubated first at 95° C.for 2 minutes and then at 42° for 5 minutes. The DNA was then sequencedby the method of Sanger et al. Proc. Natl. Acad. Sci. USA 74, 5463-6567(1977)!. The sequence analysis indicated that the correct HIV-2 env(60)gene sequence had been integrated into the BglII restriction site ofplasmid pDS78/RBSII,6xHis.

EXAMPLE 5 Reactivity of the ENV(60)-DHFR and DHFR-ENV(60) polypeptideswith HIV-positive Sera.

A. Principles

To demonstrate that the env(60) gene codes for an antigenic determinantwhich is recognized by antibodies in sera of persons infected withHIV-2, the polypeptides ENV(60)-DHFR and DHFR-ENV(60) were expressed inE. coli, transferred to a nitrocellulose filter and incubated withsuitable sera. The HIV-1 ENV(80)-DHFR polypeptide Certa et al., EMBO J.5, 3051-3056 (1986)!. was used as the control.

B. Expression of the ENV(60)-DHFR and DHFR-ENV(60) Polypeptides in E.coli

E. coli M15 cells containing plasmid pDMI,1 were transformed withplasmid penv(60)-DHFR or pDHFR-env(60) and grown in LB medium Maniatiset al., supra! containing 100 μg/ml ampicillin and 20 μg/ml kanamycin.The cultures were induced with IPTG (2 mM final conc.) at an opticaldensity of OD₆₀₀ =0.7 and grown for 4 hours. Thereafter, the cells wereharvested by centrifugation.

C. Analysis of the ENV(60)-DHFR and DHFR-ENV(60) Polypeptides Expressedin E. coli

50 μl of the cell cultures were re-suspended in 125 mM Tris-HCl, pH 6.8,containing 3% SDS, 3% β-mercaptoethanol and 20% glycerol. The sampleswere boiled for 5 minutes and subsequently separated using 12.5%polyacrylamide gel electrophoresis U.K. Laemmli, Nature 227, 680-685(1970)!. The protein bands were visualized using Coomassie blue staining(FIG. 10, upper part).

D. Reactivity of the ENV(60)-DHFR and DHFR-ENV(60) Polypeptides with HIVAntibodies

Probes of the induced cell cultures (see section B), an E. coli controllysate and purified HIV-1 ENV(80)-DHFR polypeptide (1 μg per lane) wereseparated electrophoretically (2 gels) as described in paragraph C. Eachof the unstained gels was covered with a nitrocellulose membrane. Eachof the covered gels was then covered with 2 sheets of filter paper andtransferred into a transfer chamber. This chamber was filled withtransfer buffer (25 mM Tris, pH 8.3. containing 192 mM glycine and 20%methanol) and the transfer of the polypeptides was performed at 4° C.during 12 hours and an amperage of 100 mA.

Thereafter, the nitrocellulose membranes were incubated first at roomtemperature for 4 hours in PBS Maniatis et al., supra! containing 5%dried skimmed milk. Then, one of the nitrocellulose membranes wasincubated at room temperature for 4 hours in PBS containing 5% driedskimmed milk and HIV-1-positive serum diluted 1:1000, and the othermembrane was incubated at room temperature for 4 hours in PBS containing5% dried skimmed milk and HIV-2 positive serum diluted 1:1000.Thereafter, the nitrocellulose membranes were washed first 3× with PBScontaining 0.3% Tween-20 and thereafter incubated at room temperaturefor 1 hour in PBS containing 0.3% Tween 20, 5% goat serum andgoat-anti-human-IgG-peroxidase conjugate diluted 1:2000. After threewashings with PBS, the protein bands on the nitrocellulose membraneswhich had reacted with antibodies were visualized by incubation in 25 mlof PBS containing 5 μg of 4-chloro-1-naphthol and 5 μl of 30% hydrogenperoxide. The reaction was stopped by the addition of PBS.

As shown in FIG. 10, the ENV(60)-DHFR and DHFR-ENV(60) polypeptides wererecognized only by antibodies in the serum of patients infected withHIV-2. The ENV(80)-DHFR polypeptide was recognized only by antibodies inthe serum of patients infected with HIV-1.

EXAMPLE 6 Construction of Plasmid pRBSII-6xHis

A. Description of Plasmid pDS56/RBSII

For the construction of plasmid pRBSII-6xHis, plasmid pDS56/RBSII wasused. E. coli cells transformed with this plasmid and with the plasmidpDMI,1 were deposited under the Budapest Treaty at the DeutschenSammlung von Mikroorganismen in Braunschweig on Dec. 23, 1987 E. coliM15 (pDS56/BSII: pDMI,1), DSM No.: 4330!.

The part of pDS56/RBSII (FIGS. 11 and 12) which lies between thecleavage sites for the restriction enzymes XbaI and XhoI and whichcontains the replication region and the gene for β-lactamase, whichconfers ampicillin resistance to the cells, was derived originally fromplasmid pRB322 (Bolivar et al., supra; Sutcliffe, supra). However, thegene for β-lactamase was modified by eliminating the cleavage sites forthe restriction enzymes HincII and PstI. These alterations in the DNAsequence did not, however, affect the amino acid sequence of theβ-lactamase. The remaining part of the plasmid carries the regulatablepromoter/operator element N25OPSN25OP29 followed by the ribosomalbinding site RBSII, which is part of an EcoRI/BamHI fragment. Theterminator t_(o) of the E. coli phage lambda (Schwarz et al., supra),the promoter-free gene of chloramphenicol acetyltransferase (Marcoli etal., supra) and the terminator T1 of the E. coli rrnB operon (Brosius etal., supra) follow.

Because of the high efficiency of the expression signals N25OPSN25OP29and RBSII, plasmid pDS56/RBSII and derivatives thereof such as plasmidpRBSII-6xHis can only be stably maintained in E. coli cells when thepromoter/operator element is repressed by the binding of a lac repressorto the operator (see Example 2, Section A).

B. Construction of Plasmid pRBSII-6xHis

2 pmol of plasmid pDS56/RBSII were cleaved with the restriction enzymeHindIII. After working up the sample, 50 pmol of a phosphorylatedadaptor (which codes for 6 histidines) were added to the cleaved plasmidDNA, and the sample was incubated with T4-DNA ligase as described above.After working up the ligation mixture, the DNA was cleaved with therestriction enzymes BamHI and XbaI and the BamHI/XbaI, fragmentcontaining the region coding for 6 histidines, the terminator t_(o), thecat gene and the terminator T1 was isolated by agarose gelelectrophoresis (FIG. 13).

2 pmol of plasmid pDS56/RBSII were cleaved with the restriction enzymesXbaI and BamHI, and the XbaI/BamHI fragment containing the replicationregion, the bla gene, the promoter N25OPSN25OP29 and the ribosomalbinding site RBSII was isolated by agarose gel electrophoresis (FIG.14).

0.1 pmol of each of the isolated fragments were ligated and subsequentlytransformed into E. coli strain M15 (pDMI,1) as described above (Example2). After plating and incubation (Example 2.B) individual colonies weregrown in 10 ml of medium, as described, and the plasmid DNAs wereisolated according to the method of Birnboim and Doly (supra). Arestriction analysis with the enzymes BamHI and XbaI indicated that theplasmids contained the 2 desired fragments. These plasmids weredesignated pRBSII-6xHis (FIG. 14).

EXAMPLE 7 Construction of Plasmid pRBSII-env(80)-gag(419)-env(60)-6xHis

A) Principles

For the construction of plasmid pRBSII-env(80)-gag(419)-env(60)-6xHis,the HIV-2 gene env(60) and the HIV-1 genes env(80) and gag(419) werelinked with the expression vector pRBSII-6xHis.

B) Preparation of Plasmid pRBSII-6xHis Cleaved with the RestrictionEnzymes BamHI and BglII

2 pmol of plasmid pRBSII-6xHis were cleaved with 10 units of therestriction enzymes BamHI and BglII. After adding sample buffer, the DNAwas separated in a 1% agarose gel. After staining with ethidium bromide,the band corresponding to the plasmid DNA was cut out under UV light(300 nm) and purified by electroelution (Maniatis et al., supra). Theends of the purified DNA were then dephosphorylated with phosphatase.Subsequently, the DNA was extracted with phenol:chloroform (1:1),precipitated with ethanol and dissolved in 1/4 TE buffer.

C) Preparation of the HIV-1 Gag(419) Gene

2 pmol of plasmid pU-GAG, which contains a SstI/BglII fragment of theHIV-1 gag gene (the nucleotide sequence of which is shown in FIG. 16),were digested with 12 units of the restriction enzyme XmnI.Subsequently, the DNA was extracted with phenol:chloroform (1:1) andprecipitated with 2 volumes of ethanol at -20° C. The DNA pellet wasresuspended in 50 μl of 50 mM Tris-HCl, pH 7.6, containing 10 mM MgCl₂,10 mM DTT, 0.5 mM ATP, 100 pmol phosphorylated 10 mer BamHI-linker(5'-CCGGATCCGG-3'). After adding one unit of T4-DNA ligase, the mixturewas incubated at 14° C. overnight. The mixture was then incubated at 65°C. for 10 minutes and brought to a volume of 100 μl with restrictionenzyme buffer. 100 units of BamHI and 10 units of HindIII were added,and the mixture was incubated at 37° C. for 3 hours. Subsequently, theDNA was extracted with phenol and chloroform and precipitated withethanol. The DNA pellet was dissolved in sample buffer and separated ina 6% polyacrylamide gel. After staining with ethidium bromide, a bandhaving 250 base pairs was cut out and isolated by electroelution.

2 pmol of the plasmid p2-3U/HindIII10, which contains a HindIII fragmentof the HIV-1 gag gene (the sequence of which is shown in FIG. 17), weredigested at 37° C. for one hour with 11 units of BglII, heated to 65° C.for 10 minutes, cooled to room temperature and incubated for 45 minuteswith the Klenow fragment of E. coli DNA polymerase. Thereafter, the DNAwas extracted with phenol:chloroform (1:1), precipitated with ethanol,taken up in 50 μl of 50 mM Tris-HCl, pH 7.6, containing 10 mM MgCl₂, 10mM DTT, 0.5 mM ATP, 100 pmol phosphorylated 12 mer BglII-linker(5'-GGAAGATCTTCC-3') and 1 unit of T4-DNA ligase and incubated at 14° C.overnight. The reaction mixture was then heated to 65° C. and brought toa volume of 100 μl with restriction enzyme buffer. 100 units of BglIIwere added and thereafter the mixture was incubated at 37° C. for 3hours. After adding NaCl (50 mM final concentration) 10 units of HindIIIwere added and the mixture was incubated at 37° C. for 1 hour. Afterextraction with phenol:chloroform (1:1) the DNA was precipitated withethanol and subsequently separated in a 1% agarose gel. A fragmenthaving 1020 base pairs was cut out and isolated and purified byelectroelution.

1 pmol each of the BamHI-HindIII fragment and the HindIII-BglII fragmentwere linked with each other using 1 unit of T4-DNA ligase. After heatinactivation of the ligase the ligated fragments were cleaved with BamHIand BglII and separated in a 1% agarose gel. The fragment correspondingto the HIV-1 gag(419) gene was isolated and purified by electroelution(FIG. 15).

D) Preparation of the HIV-1 Env(80) Gene

The preparation of the HIV-1 env(80) gene was carried out as describedby Certa et al. EMBO J.5, 3051-3056 (1986)!.

E) Preparation of the HIV-2 Env(60) Gene

The preparation of the HIV-2 env(60) gene is described in Example 1.

F) Construction of Plasmid pRBSII-gag(419)-6xHis

For the construction of plasmid pRBSII-gag(41g)-6xHis, 0.1 pmol of thevector pRBSII-6xHis (see B) linearized with BamHI and BglII was linkedwith 0.3 pmol of the gag(419) gene by incubation with 1 unit of T4-DNAligase at 14° C. overnight. After heat inactivation of the enzyme, theDNA was transformed into E. coli strain W3110 containing plasmid pDMI,1.The cells were plated onto LB agar plates containing 100 μg/mlampicillin and 25 μg/ml kanamycin. The plates were incubated at 37°overnight. Individual colonies were grown at 37° C. overnight, and theirplasmid DNAs were isolated using standard methods Maniatis et al.,supra! (FIG. 18).

G) Construction of Plasmid pRBSII-env(80)-gag(419)-6xHis

For the construction of plasmid pRBSII-env(80)-gag(419)-6xHis, 0.1 pmolof plasmid pRBSII-gag(419)-6xHis were cleaved with BamHI and treatedwith CIP. The DNA was extracted with phenol:chloroform (1:1) andprecipitated with 2 volumes of ethanol. The pRBSII-gag(419)-6xHisplasmid DNA linearized with BamHI was incubated at 14° C. overnight with0.3 pmol of the HIV-1 env(80) gene and 1 unit of T4-DNA ligase. Afterheat inactivation of the enzyme, the DNA was transformed into E. colistrain W3110, which contained the plasmid pDMI,1. The cells were platedonto LB agar plates containing 100 μg/ml ampicillin and 25 μg/mlkanamycin. The plates were incubated at 37° C. overnight. Individualcolonies were grown at 37° C. overnight, and their plasmid DNAs wereisolated using standard methods Maniatis et al., supra! (FIG. 19).

H) Construction of Plasmid pRBSII-env(80)-gag(419)-env(60)-6xHis

For the construction of plasmid pRBSII-env(80)-gag(419)-env(60)-6xHis,0.1 pmol of plasmid pRBSII-10 env(80)-gag(419)-6xHis was cleaved withBglII and treated with CIP. Subsequently, the DNA was extracted withphenol:chloroform (1:1) and precipitated with 2 volumes of ethanol.Plasmid pRBSII-env(80)-gag(419)-6xHis DNA linearized with BglII was thenincubated at 14° C. overnight with 0.3 pmol of the HIV-2 env(60) geneand 1 unit of T4-DNA ligase. After heat inactivation of the enzyme, theDNA was transformed into E. coli strain W3110 containing the plasmidpDMI,1. The cells were plated onto LB agar plates containing 100 μg/mlampicillin and 25 μg/ml kanamycin. The plates were incubated at 37° C.overnight. Individual colonies were grown at 37° C. overnight and theirplasmid DNAs were isolated using standard methods Maniatis et al.,supra! (FIG. 20).

EXAMPLE 8 Reactivity of the ENV(80)-GAG(419)-ENV(60) Polypeptide withHIV-Positive Sera

A. Principles

To demonstrate that the ENV(80)-GAG(419)- -ENV(60) polypeptide reactswith sera of persons infected with HIV-1 and HIV-2, theENV(80)-GAG(419)-ENV(60) polypeptide was expressed in E. coli, purifiedand tested with suitable sera in an enzyme immunoassay (EIA).

B. Expression of the ENV(80)-GAG(419)-ENV(60) Polypeptide in E. coli

E. coli W3110 cells containing plasmid pDMI,1 were transformed withplasmid pRBSII-env(80)-gag(419)-env(60)-6xHis and grown in LB mediumManiatis et al., supra! containing 100 μg/ml ampicillin and 25 μg/mlkanamycin. The culture was induced with IPTG (2 mM final conc.) at anoptical density of OD₆₀₀ =1.0 and grown for 2 hours. Thereafter, thecells were harvested by centrifugation.

C. Purification of the ENV(80)-GAG(419)-ENV(60) Polypeptide Expressed inE. coli

The harvested cells were re-suspended in PBS buffer (0.2 g/l KCl, 8.0g/l NaCl, 0.2 g/l KH₂ PO₄, 1.144 g/l Na₂ HPO₄, pH 7.0) and disruptedusing a high pressure homogenizer. The cell homogenizate obtained wascentrifuged, and the pellet was extracted twice with 0.1M sodiumphosphate buffer, pH 8.0, containing 6M guanidine.HCl.

Undissolved material was removed by centrifugation and, where required,filtered. The clarified solution was applied to a NTA column (thestructure and production of which are described in European PatentApplication Publication No. 253 303). The column was washed with 0.1Msodium phosphate buffer, pH 8.0, containing 6M guanidine.HCl and thenwith 0.1M sodium phosphate buffer, pH 6.5, containing 8M urea. Theelution of the ENV(80)-GAG(419)-ENV(60) polypeptide was carried out with0.1M sodium phosphate buffer, pH 4.0, containing 8M urea.

The ENV(80)-GAG(419)-ENV(60) obtained using the NTA column wassubsequently chromatographed twice in a Sephacryl® S-200 columnPharmacia, elution buffer: 50 mM Tris.HCl, pH 7.0, containing 5 mM EDTAand 1% SDS (first pass) or 0.1% SDS (second pass). By SDS polyacrylamidegel electrophoresis and amino acid analysis, the purifiedENV(80)-GAG(419)-ENV(60) polypeptide was shown to be 89% pure.

D. Reactivity of the Purified ENV(80)-GAG(419)-ENV(60) Polypeptide withHIV-positive Sera

The purified ENV(80)-GAG(419)-ENV(60) polypeptide was tested for itsreactivity with HIV-1 and HIV-2 positive sera in an enzyme immunoassay(EIA) as described in European Patent Application Publication No. 270114. The EIA values obtained with purified ENV(80)-GAG(419)-ENV(60)polypeptide and purified DHFR-ENV(60) polypeptide (positive HIV-2control) gave the following results:

    ______________________________________                                                                  ENV(80)-                                                          DHFR-ENV(60)                                                                              GAG(419)-ENV(60)                                    Sera          HIV-2       HIV-1/HIV-2                                         ______________________________________                                        HIV-1 positive sera.sup.1)                                                    Mil 3         0.132       1.684                                               Mil 22        0.104       2.094                                               Mil 24        0.140       2.244                                               Mil 26        0.062       2.198                                               Mil 30        0.059       2.345                                               HIV-2 positive sera.sup.1)                                                    S             1.271       2.072                                               K             0.910       2.052                                               D             1.559       2.252                                               B             1.232       2.104                                               Negative sera                                                                 N1            0.096       0.092                                               N2            0.047       1.110                                               N3            0.063       0.076                                               N4            0.056       0.067                                               N5            0.067       0.088                                               ______________________________________                                         .sup.1) confirmed by "Western blots" (WB).                               

As can be seen, all positive HIV-1 and HIV-2 sera were recognized by theENV(80)-GAG(419)-ENV(60) polypeptide.

What is claimed is:
 1. A process for detecting antibodies against HIV-2viruses in human serum, which process comprises:(a) labeling apolypeptide having the amino acid sequence ##STR2## (b) reacting thelabeled polypeptide with a human serum sample suspected of containingantibodies against HIV-2 viruses and allowing labeledpolypeptide-antibody complexes to form in the reaction mixtures; and (c)detecting the labeled polypeptide-antibody complexes in the serumsample.
 2. A process for detecting antibodies against HIV-2 viruses inhuman serum, which process comprises:(a) immobilizing on a solid supporta polypeptide having the amino acid sequence ##STR3## (b) contacting ahuman serum sample suspected of containing antibodies against HIV-2viruses with the immobilized polypeptide and allowing immobilizedpolypeptide-antibody complexes to form; (c) washing the complexes toremove unbound materials; and (d) detecting the washed complexes by theaddition of a reagent selected from the group consisting of labeledStaphylococcus aureus protein A and a labeled second anti-human Igantibody.
 3. A process for detecting HIV-2 viruses or fragments thereofin human serum or in other biological fluids, which processcomprises:(a) reacting a human serum or other biological fluid samplesuspected of containing HIV-2 viruses or fragments thereof with a knownamount of antibodies against a polypeptide having the amino acidsequence ##STR4## (b) allowing the antibodies and sample to interact toform antigen-antibody complexes in the reaction mixture; and (c)detecting the antigen-antibody complexes in the reaction mixture.
 4. Theprocess of claim 3, in which the antibodies are labeled with an enzymeand the antigen/antibody complexes are detected by an enzymeimmunoassay.